PCR & Exonuclease activity

Shiao Y. Wang sywang at whale.st.usm.edu
Tue Jun 21 10:05:15 EST 1994


I am sequencing PCR products directly. To prepare the template, one of the
primers is biotinylated at the 5' end. I then use strepavidin to
isolated the single stranded DNA for sequencing. I have been getting very
low signals lately. 

My question: could it be that the 5' -> 3' exonuclease activity of the
polymerase is removing the 5'-end of the product thereby removing the
biotin? Without the biotin, I can't capture the DNA and thus no template
for sequencing.

Most thermal stable DNA polymerase seems to have this activity. For
folks that want to do something with the DNA product (like cloning), how do
they get around this problem?

Thanks for your attention and possible reply.

Shiao Wang
University of Southern Mississippi




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